The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM. The secondary and higher order structures in short DNA repeats regions can lead to slippage, for example, by stalling the replication fork. The slippage is one of the primary reasons that lead to length polymorphism. Simulation results have predicted higher order G quadruplex structures in ILPR region. However, they have not been observed experimentally, although similar structures have been found in other G rich regions. It is known that helicase can unwind simple G-quadruplex structures, but fails to do so when these structures are stabilized by certain ligands. Based on these facts, the project proposes that there exist higher order G quadruplex structures that are recalcitrant to the helicase unwinding in the ILPR domain. Since mechanical unfolding of single biomolecules has a unique capability to reveal short-lived intermediate structures, a dual-beam dual-trap laser tweezers instrument is employed to investigate the G quadruplex and its potential higher order structures in the ILPR domain at the single molecular level. For this purpose, DNA constructs containing ILPR fragments will be synthesized using a universally applicable strategy that has been successfully developed in authors'laboratories. To identify the existence of higher order G quadruplex structures, conventional biochemical approaches will be employed. To help future development of therapeutics that targets the ILPR repeats, the interaction between G quadruplexes and various ligands, such as porphyrin and insulin, will be characterized using both single molecule and biochemical approaches. To test whether the RecQ DNA helicase is incapable of unwinding individual higher order G quadruplexes, single molecular assays will be performed using laser tweezers. Although RecQ DNA helicase and ILPR DNA fragments are used in the proposed experiments, the finding from this project will be generic enough to shed light on the cause of length polymorphism that is implicated in various diseases including IDDM, fragile X syndrome and Huntington's disease. PUBLIC HEALTH RELEVANCE The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM.